Measurement of low-abundance cytokine mRNA in cells of murine lymphoid organs: a new quantitative reverse transcription/polymerase chain reaction method
| Autor: | Erica S. Berleth, B. J. Dolnick, M. J. Ehrke, P. Ujhazy, Jane M. Meer |
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| Rok vydání: | 1999 |
| Předmět: |
Quality Control
Cancer Research Lymphoid Tissue medicine.medical_treatment Immunology Thymus Gland Mice Immune system Gene expression medicine Immunology and Allergy Animals Ascitic Fluid RNA Messenger Cells Cultured biology Reverse Transcriptase Polymerase Chain Reaction Tumor Necrosis Factor-alpha Reproducibility of Results Molecular biology Reverse transcription polymerase chain reaction Mice Inbred C57BL Real-time polymerase chain reaction Cytokine Oncology Cell culture Concanavalin A biology.protein Cytokines Tumor necrosis factor alpha Female Spleen Interleukin-1 |
| Zdroj: | Cancer immunology, immunotherapy : CII. 48(8) |
| ISSN: | 0340-7004 |
| Popis: | To investigate cytokine regulation in cells of freshly excised lymphoid tissues, rigorous quantitative reverse transcription/polymerase chain reaction (QRT-PCR) assays were developed to measure attomole (10(-18) mol) amounts of the mRNA for seven cytokines: interleukin-1alpha (IL-1alpha), IL-1beta, tumor necrosis factor alpha (TNFalpha), interferon gamma (IFNgamma), transforming growth factor beta (TGFbeta), IL-2 and IL-6. RNA was purified from single-cell suspensions of immune tissues (spleen, thymus and resident peritoneal cells). Data are presented demonstrating the utility of these assays for quantifying basal levels of all seven cytokine mRNAs in the freshly isolated splenocytes and thymocytes. Studies to establish the usefulness of these assays for measuring changes in the levels of cytokine mRNA focused on IL-1alpha, IL-1beta, TNFalpha and IL-2 in splenocytes, thymocytes and resident peritoneal cells. Using the QRT-PCR assays developed, levels of cytokine mRNA could be quantified in RNA samples obtained both from freshly isolated cells and from cells following short-term (/=26 h) culture. These measurements established basal in vivo levels of specific cytokine mRNAs and demonstrated specific modulation of their levels by cell culture and by the inclusion of immune stimulants (bacterial lipopolysaccharide or the plant lectin concanavalin A) in the culture. These data provide new information on both basal and stimulated cytokine levels that is required for valid interpretations of the roles of cytokine expression in immune regulation. |
| Databáze: | OpenAIRE |
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